title: Spatial calibration of structured illumination fluorescence microscopy using capillary tissue phantoms.
abstract: Quantitative assessment of microvascular structure is relevant to the investigations of ischemic injury, reparative angiogenesis and tumor revascularization. In light microscopy applications, thick tissue specimens are necessary to characterize microvascular networks; however, thick tissue leads to image distortions due to out-of-focus light. Structured illumination confocal microscopy is an optical sectioning technique that improves contrast and resolution by using a grid pattern to identify the plane-of-focus within the specimen. Because structured illumination can be applied to wide-field (nonscanning) microscopes, the microcirculation can be studied by sequential intravital and confocal microscopy. To assess the application of structured illumination confocal microscopy to microvessel imaging, we studied cell-sized microspheres and fused silica microcapillary tissue phantoms. As expected, structured illumination produced highly accurate images in the lateral (X-Y) plane, but demonstrated a loss of resolution in the Z-Y plane. Because the magnitude of Z-axis distortion was variable in complex tissues, the silica microcapillaries were used as spatial calibration standards. Morphometric parameters, such as shape factor, were used to empirically optimize Z-axis software compression. We conclude that the silica microcapillaries provide a useful tissue phantom for in vitro studies as well as spatial calibration standard for in vivo morphometry of the microcirculation.
authors.affiliation: Laboratory of Adaptive and Regenerative Biology, Brigham and Women's Hospital, Department of Surgery, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Grace S
authors.initials: GS
authors.lastname: Lee
authors.forename: Lino F
authors.initials: LF
authors.lastname: Miele
authors.forename: Aslihan
authors.initials: A
authors.lastname: Turhan
authors.forename: Miao
authors.initials: M
authors.lastname: Lin
authors.forename: Dusan
authors.initials: D
authors.lastname: Hanidziar
authors.forename: Moritz A
authors.initials: MA
authors.lastname: Konerding
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Mentzer
mesh_terms: D000818:Animals; D002138:Calibration; D002196:Capillaries / Q000648:ultrastructure*; D002232:Carbocyanines / Q000737:chemistry; D003627:Data Interpretation, Statistical; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008833:Microcirculation; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008863:Microspheres; D055806:Microvessels / Q000648:ultrastructure*; D017711:Nonlinear Dynamics; D019047:Phantoms, Imaging; D012822:Silicon Dioxide / Q000737:chemistry; D012984:Software
chemical_list: D002232:Carbocyanines; C024286:3,3'-dioctadecylindocarbocyanine; C098044:3,3'-dioctadecyloxacarbocyanine; D012822:Silicon Dioxide